Punches were blocked in IF buffer (1% BSA and 0.05% Tween‐20 in PBS) for 1–2 h at RT, followed by the incubation with primary antibody diluted in IF buffer, for 24 h at 4°C. Punches were washed in 1× PBS for 1–2 h and incubated for 24 h at 4°C with secondary antibody and DAPI (ThermoFisher; D1306) diluted 1:10 000 in IF buffer. The following primary antibodies were used for immunostaining of expanded samples: mouse anti‐acetylated tubulin (Sigma, T7451) at 1:4000, mouse anti‐alpha tubulin (Sigma; T6074) at 1:500, rabbit antibeta tubulin (Abcam; ab15568) at 1:250, rabbit anti‐ARL13B (Abcam; ab83879) at 1:150, rabbit anti‐Cep164 (Proteintech; 22227‐1‐AP) at 1:500, rabbit anti‐ANKRD26 (GeneTex; GTX128255) at 1:200, rabbit anti‐FBF1 (sigma; HPA023677) at 1:100, rabbit anti‐RPGRIP1L (Proteintech; 55160‐1‐AP) at 1:150, rabbit anti‐IFT88 (Proteintech; 13967‐1‐AP) at 1:150, mouse anti‐Rootletin (Santa Cruz; sc‐374056) at 1:50, rabbit antipericentrin (Abcam; ab4448) at 1:400, rabbit anti‐Cep290 (Abcam; ab84870) at 1:150 and rabbit antipolyglutamylated tubulin (AdipoGen; AG‐25B‐0030) at 1:800. Secondary antibodies antimouse Alexa Fluor 488 (Invitrogen; A11029), antirabbit Alexa Fluor 488 (Invitrogen; A11034), antimouse Alexa Fluor 555 (Invitrogen; A28180), antirabbit Alexa Fluor 555 (Invitrogen; A21429) were used at a 1:800 dilution to label primary antibodies.
A28180
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A28180 is a laboratory instrument designed for the measurement and analysis of various samples. It is a compact and versatile device that can be used for a wide range of applications within the scientific research and testing fields.
Automatically generated - may contain errors
Lab products found in correlation
Ab15568, by Abcam (1 mentions)
Ab4448, by Abcam (1 mentions)
Ab84870, by Abcam (1 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
T7451, by Merck Group (1 mentions)
T6074, by Merck Group (1 mentions)
Ag 25b 0030, by Adipogen (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
A21429, by Thermo Fisher Scientific (1 mentions)
Laser confocal microscope, by Leica Microsystems (1 mentions)
Ab49873, by Abcam (1 mentions)
Tissue tek, by Ted Pella (1 mentions)
Ab104139, by Abcam (1 mentions)
A 11015, by Thermo Fisher Scientific (1 mentions)
Sc 5274, by Santa Cruz Biotechnology (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
3 protocols using a28180
1
Immunostaining of Expanded Samples
2
Immunohistochemical Localization of SHP-1, pJNK, and GS
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The eyecups were fixed in 4% paraformaldehyde for 1 hour and dehydrated sequentially in 20% and 30% sucrose solutions at 4°C for 1 hour. After the anterior sections had been removed and the cups filled with optimal cutting temperature compound (Tissue‐Tek; Ted Pella), they were then frozen at −80°C, and cut in the sagittal direction (8‐μm‐ thick sections). After being blocked with 5% goat serum and permeated with 0.15% Triton X‐100 in PBS for 45 minutes, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti‐SHP‐1(sc‐7289, diluted 1:50; Santa Cruz Biotechnology); mouse anti‐pJNK (9255S, diluted 1:100; Cell Signaling Technology) and rabbit anti‐ glutamate synthase (GS) (ab49873, diluted 1:5000; Abcam). The sections were rinsed three times with PBS and incubated with a secondary antibody (A28180; Invitrogen; or 4414, Cell Signaling Technology) for 1 hour at room temperature. After the sections were rinsed three times, they were counterstained with Fluoroshield with DAPI mounting medium (ab104139; Abcam). The sections were then observed with a laser confocal microscope (Leica Microsystems).The immunofluorescence intensity in the images was measured with ImageJ software (National Institutes of Health).
3
Flow Cytometry Analysis of Cell Differentiation
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Flow cytometry was performed to determine the differentiation efficiency. The cells were treated with trypsin for 2–5 min and harvested, followed by 400× g centrifugation for 5 min. The supernatant was removed, and the cells were resuspended to 1 × 106 cells/mL in ice cold DPBS, 0.1% BSA.
Primary antibody staining was then applied on the cells according to the manufacturer’s instructions. The washing steps were performed 3 times by centrifuging at 400× g for 5 min and resuspending them in ice-cold DPBS. After the washing steps, the cells were resuspended with diluted (according to the manufacturer’s instructions) fluorochrome-labeled secondary antibody in 0.1% BSA/DPBS for 1 h in dark, and followed by the 3 times washing steps mentioned above. Finally, all samples were acquired and analyzed on FC500 (Beckman, Brea, CA, USA).
The primary and fluorescence-conjugated secondary antibodies used in this study were as follows: KIRREL (1:100; AF2564, R&D), β-tubulin III (1:300; sc5274, Santa Cruz), anti-sheep Alexa488 (1:2000; A-11015, Invitrogen), anti-mouse Alexa555 (1:2000; A28180, Invitrogen), anti-rabbit Alexa488 (1:2000; A-11008, Invitrogen).
Primary antibody staining was then applied on the cells according to the manufacturer’s instructions. The washing steps were performed 3 times by centrifuging at 400× g for 5 min and resuspending them in ice-cold DPBS. After the washing steps, the cells were resuspended with diluted (according to the manufacturer’s instructions) fluorochrome-labeled secondary antibody in 0.1% BSA/DPBS for 1 h in dark, and followed by the 3 times washing steps mentioned above. Finally, all samples were acquired and analyzed on FC500 (Beckman, Brea, CA, USA).
The primary and fluorescence-conjugated secondary antibodies used in this study were as follows: KIRREL (1:100; AF2564, R&D), β-tubulin III (1:300; sc5274, Santa Cruz), anti-sheep Alexa488 (1:2000; A-11015, Invitrogen), anti-mouse Alexa555 (1:2000; A28180, Invitrogen), anti-rabbit Alexa488 (1:2000; A-11008, Invitrogen).
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